Animals have evolved various types of chemosensory tools to perceive the outside world. Among these are the taste and the olfactory systems. Specialized chemosensors are expressed in these structures. Depending on the species, these may include for example G protein-coupled receptors (GPCRs) such as odorant receptors (ORs), vomeronasal receptors (VRs), trace amine receptors (TAARs), formyl peptide receptors (FPRs), T1R and T2R taste receptors, and non-GPCRs such as transient receptor potential (Trp) channels, guanylyl cyclases, and olfactory ionotropic receptors (IRs) (Kaupp, 2010, Nature Rev. Neuroscience, 11: 188-200). These sensors allow the animals to face the immense variety of external stimuli, in particular via their ORs, which allows them to detect and discriminate billions of different molecules. The gene repertoire encoding these receptors is diverse inside a given species, and is variable between species, both in terms of size and in terms of contents. In the mouse, for example, the OR repertoire reaches 1'250 members, which represents over 5% of its total number of genes. Every olfactory sensory neuron expresses a single olfactory receptor gene, which means that hundreds of functionally different populations of sensory neurons coexist in the nasal cavity. Each of these populations can be activated by various agonists, and each agonist can be recognized by various ORs. This leads to a combinatorial code, which allows discrimination between different blends.
In the last 30 years, in a process called deorphanization, ligands and antagonists have been assigned to a large fraction of GPCRs. This is to the exception of ORs, which remain orphans for their largest part. For example, over 90% of human ORs are still orphans (Peterlin et al., 2014, J. Gen. Physiol., 143, 527-542). Individual OR deorphanization is important, but even more interesting would be to provide a list of ORs that respond to a given chemical in a given species. Today, in mice or humans, not a single odorant molecule is known for which an exhaustive list of cognate ORs has been defined. Such knowledge could be very valuable for understanding the combinatorial code at the base of the sense of smell, but could also have commercial applications. For example, the knowledge of the OR repertoire activated by a given odorant would be helpful for mimicking given olfactory stimuli, particularly those with positive hedonic values in humans (like chocolate or flowers). Alternatively this could allow the discovery of odorant antagonists blocking the perception of undesired odors or flavors (like unpleasant body odors or the smell of sewers). Taken as a whole, it would facilitate our ability to modulate specific chemosensory percepts.
The limited number of deorphanized ORs, to date, does not result from a lack of efforts, but rather from a lack of suitable assays. Most known olfactory agonist-receptor pairs were identified in vitro. These approaches involved the expression of rodent or human chemoreceptors in heterologous systems, including xenopus oocytes, yeasts, ovarian insect cells, baculoviruses, and native or engineered HEK and Hela cells (Peterlin et al., 2014, supra). These expression systems brought significant advances and allowed the deorphanization (that is to find at least one activating molecule) and characterization of 41 human and 95 mouse ORs. However, these non-native methodologies suffer from significant downsides. First, ORs produced in vitro are usually retained in the endoplasmic reticulum and thus fail to reach the cell membrane. Their fusion with segments of non-olfactory proteins is therefore often chosen for heterologous expression, possibly modifying their response profiles. Second, a complex nasal mucus containing odorant binding proteins, that represents the natural interface between receptors and their potential agonists, is absent in vitro. This is critical since this mucus plays an enzymatic role that chemically modifies many odorant molecules (Nagashima and Touhara, 2010, J. Neurosci., 30, 16391-16398). Third, the coupling of the OR to its native transduction cascade, which is usually not recapitulated in vitro, is known to affect receptor-odorant specificities (Shirokova et al., 2005, J. Biol. Chem., 280, 11807-11815). Finally, potential ligands are provided in liquid and not gaseous phase in vitro, making their concentrations difficult to relate to those present during natural ortho- and retronasal fluxes.
To circumvent some of the non-native downsides of heterologous expression, alternative approaches were taken. Efforts to develop in silico models have been made (e.g. Bavan et al., 2014, PLoS One 9, e92064). Closer to physiological conditions, responses of olfactory sensory neurons expressing endogenous or exogenous ORs to chemicals were studied (Araneda et al., 2000, Nat. Neurosci. 3, 1248-1255; Malnic et al., 1999, Cell 96, 713-723; Oka et al., 2006, Neuron, 52, 857-869). Other methods based on gene-targeted mice in which defined olfactory sensory neurons were labeled, also proved successful for a handful of ORs. However, these methodologies based on sensory neurons involve ex vivo preparations or complex mouse surgeries, and most importantly, only allow deorphanization of one receptor at a time.
Therefore, there remains a need for a method allowing the rapid and easy identification of the receptors that respond to specific olfactory compounds in vivo, in particular those of special interest like malodor counteracting molecules or smell modulators. Such a method would also constitute a critical tool for large scale screening of agonists and antagonists.